CN114957300A - Near-infrared two-region fluorescent probe for detecting hydrogen peroxide and preparation method and application thereof - Google Patents
Near-infrared two-region fluorescent probe for detecting hydrogen peroxide and preparation method and application thereof Download PDFInfo
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- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 title claims abstract description 180
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- 238000002360 preparation method Methods 0.000 title abstract description 9
- 239000000523 sample Substances 0.000 claims abstract description 59
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- -1 triethylene glycol monomethyl ether benzindole Chemical compound 0.000 claims abstract description 5
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- 238000000034 method Methods 0.000 claims description 17
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- C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
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- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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Abstract
The invention discloses a near-infrared two-region fluorescent probe for detecting hydrogen peroxide, and a preparation method and application thereof. The fluorescent probe takes phenylboronic acid amido as a response group of hydrogen peroxide and a heptamethine cyanine dye containing two triethylene glycol monomethyl ether benzindole as a fluorophore. The probe emits 900-. Two triethylene glycol monomethyl ether chains in the probe endow the probe with good water solubility, so that the probe can realize a monomolecular dispersion state in an aqueous medium, and therefore the probe can be applied to detection of hydrogen peroxide in aqueous food.
Description
Technical Field
The invention belongs to the field of analysis and detection of hydrogen peroxide in food, and particularly relates to a near-infrared two-region fluorescent probe for detecting hydrogen peroxide, and a preparation method and application thereof.
Background
Hydrogen peroxide (H) 2 O 2 ) Is a common chemical reagent, and the aqueous solution of the chemical reagent is commonly called hydrogen peroxide. Due to the strong oxidizing properties of hydrogen peroxide itselfChemical properties, such as sterilization, disinfection, bleaching, antisepsis, etc., therefore, the hydrogen peroxide has great application value in the food industry. Currently, many enterprises in various countries use food grade hydrogen peroxide to disinfect food. The hydrogen peroxide has the advantages of good disinfection effect, high efficiency, no need of cleaning, no pollution to the environment and the like. At present, the food-grade hydrogen peroxide is mainly applied to the production of food such as dairy products, aquatic products, foamed products, beer and the like, and not only comprises the disinfection of the food, but also comprises the disinfection of food packaging, production equipment and the like.
However, although hydrogen peroxide is decomposed into oxygen and water by itself, the decomposition process is affected by temperature, time, and environmental factors. Excessive residual hydrogen peroxide can not only damage the nutritional value of food itself, but also cause significant harm to human health, causing serious gastrointestinal and respiratory problems, and even death. Therefore, hydrogen peroxide is used in food production by adding and using it strictly as specified. For example, bleaching chicken feet, cattle feet, pig feet, etc. with high concentration hydrogen peroxide, firstly, prolongs the shelf life of the food, and secondly, gives the food a better appearance. Therefore, it is important to develop a simple, convenient, fast and accurate method for detecting the residual amount of hydrogen peroxide in food.
The iodometry method adopts a titration method for testing, the method has the advantages of complex operation, easy influence of human operation factors and low accuracy, and the titanium salt colorimetric method also needs complex operation steps. Other detection methods such as electrochemical method, high performance liquid chromatography, spectrophotometry, mass spectrometry and the like generally have the defects of complex operation, high cost and the like. The fluorescence probe method has the advantages of simple operation, low cost, rapidness, sensitivity and the like, thereby receiving more and more attention. Currently, some fluorescent probes have been developed for detecting hydrogen peroxide. For example, a ratio-type fluorescent probe which takes coumarin and naphthalimide as fluorophores and takes phenylboronic acid pinacol ester as a response group is developed by a fluorescent probe for detecting hydrogen peroxide, a synthetic method and application. When the probe is not reacted with hydrogen peroxide, the emission peak of coumarin appears at 473nm under the excitation of 400 nm; after the hydrogen peroxide is added, intramolecular charge transfer and fluorescence resonance energy transfer occur, and the emission peak of the probe appears at 554 nm. However, the emission wavelength of the probe is too short, the coumarin and the naphthalimide are rigid molecules with large conjugated structures, and no other hydrophilic groups exist in the molecules, so that the probe is poor in water solubility and is difficult to be applied to detection and monitoring of hydrogen peroxide in water-containing food. A benzindole-type hydrogen peroxide fluorescent probe and a preparation method thereof develop a hydrogen peroxide fluorescent probe with a hemicyanine structure, which is formed by condensing benzindole derivatives and 4-formylphenylboronic acid pinacol ester, and after the hydrogen peroxide is reacted, the fluorescence of the probe at the position of 619nm is enhanced, thereby realizing the detection of the hydrogen peroxide. However, the probe also lacks a hydrophilic group, is difficult to apply to aqueous foods, and has an emission wavelength shorter in the visible region. The synthesis and application of a fast-response hydrogen peroxide long-wavelength fluorescent probe disclose that a benzindole hemicyanine fluorescent probe is developed by using pinacol phenylboronate as a response group, the fluorescent intensity of the probe at 723nm after the response to hydrogen peroxide is greatly enhanced, and the fluorescent probe is applied to the test of chicken feet, dried tofu, pigskin and other foods; the absorption and emission wavelengths of the probe are also relatively short and do not reach the near infrared region, which limits its application to some extent. The fluorescent probe with the near-infrared two-region wavelength has more excellent performance, because in the wavelength range, the interference of autofluorescence of protein, cellulose and the like in food can be weakened more, the detection accuracy is improved, and the detection depth of a sample can be increased, so that the residual quantity of hydrogen peroxide outside and inside the food can be detected simultaneously. Therefore, the preparation of the fluorescent probe with good water solubility and near-infrared two-region wavelength has important significance for simply, conveniently, quickly and accurately detecting the residual hydrogen peroxide in the water-containing food.
Disclosure of Invention
In order to overcome the drawbacks and deficiencies of the prior art, the present invention provides a near-infrared two-zone fluorescent probe for detecting hydrogen peroxide, and a preparation method and an application thereof. In particular to a near-infrared two-region fluorescent probe capable of detecting hydrogen peroxide, a preparation method of the probe and application of the probe in food detection. The fluorescent probe takes a heptamethine cyanine dye containing two triethylene glycol monomethyl ether benzindole as a fluorophore, and the long conjugation degree enables the probe to emit 900-1150nm near-infrared two-zone fluorescence after the reaction with hydrogen peroxide, so that the problem of fluorescence interference of the probe with short wavelength is solved; in addition, triethylene glycol monomethyl ether on two sides of the probe can effectively improve water solubility, and solves the problems that other probes have poor water solubility and are difficult to exist in a monomolecular dispersion state in water-containing food.
The purpose of the invention is realized by the following technical scheme:
a near-infrared two-region fluorescent probe for detecting hydrogen peroxide, wherein the molecular formula of the probe is C 64 H 81 BN 3 O 9 + The concrete structure is as follows:
the invention provides a preparation method of a near-infrared two-zone fluorescent probe for detecting hydrogen peroxide, which comprises the following steps:
dissolving a compound Cy-NH shown in the following formula in tetrahydrofuran, adding N, N-diisopropylethylamine, introducing inert gas to enable a reaction system to be in an inert atmosphere, stirring in an ice water bath, adding 4-formyl chlorobenzene boric acid for reaction, and after the reaction is finished, separating and purifying to obtain the fluorescent probe.
Preferably, the molar ratio of the compound Cy-NH to N, N-diisopropylethylamine is 1: (4-5).
Preferably, the molar ratio of the compound Cy-NH to 4-formylchlorobenzeneboronic acid is 1: (2.5-3).
Preferably, the amount of added tetrahydrofuran per mmol of compound Cy-NH is 15-20 mL.
Preferably, the inert atmosphere is formed by vacuumizing and filling nitrogen into the system, and repeating for at least three times, wherein the inert gas is nitrogen.
Preferably, the stirring time under the ice-water bath is 10 to 20 minutes.
Preferably, the reaction is carried out at room temperature for 12-15 h.
Preferably, the method of purification is silica gel chromatography.
Preferably, the eluent used for silica gel chromatography is dichloromethane/methanol.
The invention also provides application of the near-infrared two-region fluorescent probe for detecting hydrogen peroxide in food.
Further, the application of the near-infrared two-region fluorescent probe for detecting hydrogen peroxide in the detection of hydrogen peroxide in the water-containing food.
Compared with the prior art, the fluorescent probe provided by the invention has the outstanding advantages that:
(1) the fluorescent probe takes the phenylboronic acid amide group as a response group, and has a specific recognition function on hydrogen peroxide; the heptamethine cyanine dye containing two triethylene glycol monomethyl ether benzindole is a fluorophore and has long conjugation degree, and the probe can emit near-infrared two-region fluorescence of 900-1150nm after responding to hydrogen peroxide. Compared with other probes with short wavelength, the probe can effectively reduce interference of autofluorescence caused by protein, cellulose and the like. In addition, because the light is emitted in the near infrared region II, the detection depth is greatly increased, and therefore the residual hydrogen peroxide outside and inside the food can be detected simultaneously.
(2) The indole onium salts at two ends of the fluorescent probe are respectively modified with triethylene glycol monomethyl ether. Compared with a probe without a hydrophilic group or a single triethylene glycol monomethyl ether, the water solubility of the probe is remarkably improved, and the probe can form hydrogen bond association with hydroxyl contained in the probe when being applied to food detection, so that a monomolecular dispersion state is realized, and the phenomenon of fluorescence quenching caused by the aggregation of the probe in an aqueous solution is avoided. Therefore, the fluorescent probe of the present invention is suitable for detection of water-containing foods.
(3) The fluorescent probe has strong anti-interference capability, does not respond to ions, amino acids and the like in food, and has good specificity on hydrogen peroxide. And the lower limit of detection is as low as 0.66 mu M, which is far lower than the minimum residual quantity of hydrogen peroxide specified in food. Therefore, when the probe is applied to food detection, the operation is simple, the speed is high, the accuracy is high, and the reliability is high.
(4) When the fluorescent probe reacts with hydrogen peroxide, the fluorescence intensity at the peak 930nm has a good linear relation with the concentration of the hydrogen peroxide, and the linear regression equation is that Y is 9.15968X +276.7466, R 2 0.997, so the probe can quantitatively detect the hydrogen peroxide in the food.
Drawings
FIG. 1 is a scheme showing the synthesis of the fluorescent probe of the present invention.
FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of the fluorescent probe in example 1.
FIG. 3 is a high resolution mass spectrum of the fluorescent probe of example 1.
FIG. 4 is a graph showing fluorescence emission spectra measured by adding hydrogen peroxide at various concentrations to the probe in application example 1.
FIG. 5 is a linear graph of fluorescence intensity at 930nm measured in application example 1 as a function of hydrogen peroxide concentration.
FIG. 6 is a graph showing the change of fluorescence intensity with time, which was measured by adding hydrogen peroxide to the probe in application example 2.
FIG. 7 is a graph showing the relationship between the fluorescence intensity at 930nm and the time, which was measured by adding hydrogen peroxide to the probe in application example 2.
FIG. 8 is a graph showing the selectivity of the fluorescent probe used in example 3.
Detailed Description
The following description of the embodiments of the present invention is provided in connection with the accompanying drawings and examples, but the invention is not limited thereto. It is noted that the processes described below, if not specifically described in detail, are all realizable or understandable by those skilled in the art with reference to the prior art. The reagents or apparatus used are not indicated to the manufacturer, and are considered to be conventional products available by commercial purchase.
The synthetic route of the near-infrared two-zone fluorescent probe for detecting hydrogen peroxide is shown in figure 1.
Example 1
224.81mg (0.25mmol) of the compound Cy-NH was taken in a 100mL two-necked flask, 3.75mL of tetrahydrofuran was added thereto and dissolved, 165. mu.L (1mmol) of N, N-diisopropylethylamine was further added thereto, nitrogen was introduced thereto to put the reaction system under a nitrogen atmosphere, stirring was carried out in an ice water bath for 10 minutes, 115.24mg (0.625mmol) of 4-formylchlorobenzeneboronic acid was added thereto and then reacted for 12 hours. The tetrahydrofuran was removed by rotary evaporation, and the resulting solid was purified by silica gel column chromatography (eluent dichloromethane/methanol, V/V ═ 2: 1) to obtain 80mg of a fluorescent probe (yield: 30.56%).
It was characterized by means of nuclear magnetic resonance hydrogen spectroscopy: 1 H NMR(600MHz,CD 3 OD) δ 8.30-8.24 (m,2H),8.00 (t, J ═ 7.6Hz,4H),7.87(dd, J ═ 14.1,6.1Hz,2H), 7.69-7.48 (m,10H),6.41(d, J ═ 14.2Hz,2H), 4.51(t, J ═ 5.2Hz,4H),3.97(t, J ═ 6.6Hz,4H), 3.61-3.56 (m,7H), 3.52-3.47 (m,4H),3.39(d, J ═ 5.1Hz,4H), 3.28-3.25 (m,4H),3.17(s,6 dd), 2.87 (J ═ 90.9,15.9, 2H), 2.06-2.31, 2.25 (m,4H),3.17(s,6 ddh), 2.87 (J ═ 90.9,15.9, 2H), 2.06(m, 1H), 2.05, 1H), 1H), 3.9, 1H, 8.07 (d, 1H). The NMR spectrum is shown in FIG. 2.
It was further verified by high resolution mass spectrometry: HR-MS (ESI, m/z): theoretical calculation of molecular mass to charge ratio C 64 H 81 BN 3 O 9 + [M] + : 1046.6060, the actual molecular mass to charge ratio is: 1046.6071. the high resolution mass spectrum is shown in figure 3.
Example 2
449.63mg (0.5mmol) of the compound Cy-NH was put in a 100mL two-necked flask, 9mL of tetrahydrofuran was added thereto and dissolved, and then 372. mu.L (2.25mmol) of N, N-diisopropylethylamine was added thereto, nitrogen was introduced thereto to keep the reaction system under a nitrogen atmosphere, and after stirring for 15 minutes in an ice water bath, 248.91mg (1.35mmol) of 4-formylchlorobenzeneboronic acid was added and reacted for 14 hours. The tetrahydrofuran was removed by rotary evaporation, and the resulting solid was purified by silica gel column chromatography (eluent dichloromethane/methanol, V/V ═ 2: 1) to obtain 150mg of a fluorescent probe (yield: 28.65%).
The fluorescent probe obtained in this example was the same as the characterization result in example 1.
Example 3
899.25mg (1mmol) of the compound Cy-NH was taken in a 100mL two-necked flask, 20mL of tetrahydrofuran was added and dissolved, then 826. mu.L (5mmol) of N, N-diisopropylethylamine was added, nitrogen was introduced to stir the reaction system under a nitrogen atmosphere in an ice-water bath for 20 minutes, 553.14mg (3mmol) of 4-formylchlorobenzeneboronic acid was added and the reaction was carried out for 15 hours. The tetrahydrofuran was removed by rotary evaporation, and the resulting solid was purified by silica gel column chromatography (eluent dichloromethane/methanol, V/V ═ 2: 1) to obtain 280mg of a fluorescent probe (yield: 26.74%).
The fluorescent probe obtained in this example was the same as the characterization result in example 1.
Application example 1
Adding hydrogen peroxide with different concentrations into the probe to test a fluorescence emission spectrogram:
the probe prepared in example 1 was dissolved in DMSO to prepare a test stock solution having a probe concentration of 1 mM. mu.L of the probe stock solution was added to a centrifuge tube, then, different amounts of hydrogen peroxide solutions (final concentrations of hydrogen peroxide were 0, 20. mu.M, 40. mu.M, 60. mu.M, 80. mu.M, 100. mu.M, 120. mu.M, 150. mu.M) were added, and finally, PBS buffer (pH 7.4) was added. The total volume of the test system was 3mL, and the probe concentration was 10. mu. M, DMSO at 1% by volume. As shown in FIG. 4, it can be seen from FIG. 4 that the fluorescence of the probe is relatively weak when no hydrogen peroxide is added, and the fluorescence of the test system in the range of 900-1150nm (with a peak of 930nm) is gradually enhanced with the increase of the concentration of hydrogen peroxide. And linearly fitting the relation between the hydrogen peroxide concentration and the fluorescence intensity at 930nm to obtain a fitted curve Y-9.15968X +276.7466, R 2 0.997 as shown in fig. 5. Calculating the lowest detection limit of the probe according to a calculation formula LOD of 3 sigma/K (LOD is the lowest detection limit, sigma is the standard deviation of a blank sample (namely a probe solution with the hydrogen peroxide concentration of 0 mu M) measured for multiple times (more than or equal to 20 times), and K is the slope of a fitting curve), and calculating to obtain the lowest detection limit of the probeIt was 0.66. mu.M.
Application example 2
Time test of probe response to hydrogen peroxide:
30 μ L of the probe stock solution of application example 1 was added to a centrifuge tube, followed by addition of hydrogen peroxide solution (final concentration of hydrogen peroxide solution is 150 μ M), and finally addition of PBS buffer (pH 7.4), and the total amount of the test system was 3 mL. The test is carried out by respectively incubating for 1min, 3min, 5min, 10min, 15min, 20min, 25min and 30min at room temperature. As shown in FIGS. 6 and 7, it can be seen from FIG. 6 that the fluorescence intensity of the test system in the range of 900-1150nm (with a peak of 930nm) gradually increases with time, and the increasing speed gradually slows down and hardly changes after 25 min. As can be seen from FIG. 7, the fluorescence intensity at 930nm gradually increased with time, and gradually became gentle from the beginning of rapid increase and stabilized after 25min, indicating that the response time of the probe to hydrogen peroxide was 25 min.
Application example 3
Selectivity test of probe to hydrogen peroxide:
30 μ L of the probe stock from application example 1 was added to a centrifuge tube, and the following analytes were added: a. blank (i.e., probe solution without any analyte), b.H 2 O 2 (150. mu.M), c.glutathione (1mM), d.cysteine (1mM), e.L-isoleucine (1mM), f.glutamic acid (1mM), g.tyrosine (1mM), h.alanine (1mM), i.glucose (1mM), j.arginine (1mM), k.SO 3 2- (1mM)，l.NaHS(1mM)，m.Na + (1mM)，n.K + (1mM)， o.Ca 2+ (1mM)，p.Mg 2+ (1mM) (5mM stock solution prepared for each analyte using PBS buffer (pH 7.4) as solvent, the concentration of each analyte in parentheses is the final concentration of each analyte in the test system, and the ionic analyte is Na, respectively 2 SO 3 、NaNO 3 、KNO 3 、CaCl 2 、MgCl 2 . ) Finally, PBS buffer (pH 7.4) was added, and the total amount of the test system was 3 mL. The test was performed by incubation at room temperature for 25 min. The results are shown in FIG. 8(F is the fluorescence intensity at 930nm for each groupDegree F 0 The fluorescence intensity at 930nm of the blank control group), it can be seen from the figure that the fluorescence intensity of the test group added with various other substances has no obvious change, and the fluorescence intensity of the test group added with hydrogen peroxide is obviously enhanced, and the experimental result shows that the probe has good selectivity to hydrogen peroxide.
Application example 4
The application of the probe in detecting hydrogen peroxide in food comprises the following steps:
weighing four parts of shredded squid, bamboo shoots and chicken feet, wherein each part is 1.0g, two parts are used as blank groups (without adding hydrogen peroxide), and the other two parts are used as standard groups (with adding hydrogen peroxide standard solution). Preparing a 50mM hydrogen peroxide standard aqueous solution, uniformly coating 50 mu L of the hydrogen peroxide standard aqueous solution on the surface of a food sample, placing the food sample in a shade place, placing the sample into 5mL of pure water after the sample is volatilized to be dried at room temperature, carrying out ultrasonic oscillation for 30min, taking supernatant, and centrifuging by using a high-speed centrifuge to obtain the food test solution containing the hydrogen peroxide. 30 μ L of the probe stock solution in application example 1 was added to a centrifuge tube, 300 μ L of the food test solution containing hydrogen peroxide was added, and PBS buffer (pH 7.4) was added to the centrifuge tube, so that the total amount of the test system was 3mL, and the theoretical test concentration of hydrogen peroxide was 0.05 mM. The fluorescence intensity at 930nm was collected for each test group and the concentration of hydrogen peroxide in that group was calculated according to the linear regression equation Y-9.15968X + 276.7466. The test results are shown in table 1 (test experiment results of the probe on the hydrogen peroxide concentration in food (squid silk, bamboo shoots and chicken paws)), and it can be seen that the concentration of hydrogen peroxide in the standard group measured by the fluorescence detection method is similar to that of the added hydrogen peroxide standard solution, and the standard recovery rate is within a reasonable range, so that the probe has higher accuracy in detecting the hydrogen peroxide content in the food.
TABLE 1 concentration of hydrogen peroxide in food products
The above examples are preferred embodiments of the present invention, but the present invention is not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and they are included in the scope of the present invention.
Claims (10)
1. A near-infrared two-region fluorescent probe for detecting hydrogen peroxide is characterized in that the molecular formula of the probe is C 64 H 81 BN 3 O 9 + Having the formula:
2. the method for preparing the near-infrared two-zone fluorescent probe for detecting hydrogen peroxide as claimed in claim 1, characterized by comprising the following steps:
dissolving a compound Cy-NH shown in the specification in tetrahydrofuran, adding N, N-diisopropylethylamine, introducing inert gas to enable a reaction system to be in an inert atmosphere, stirring in an ice water bath, adding 4-formyl chlorobenzene boric acid to react, and after the reaction is finished, separating and purifying to obtain the fluorescent probe.
3. The method for preparing a near-infrared two-zone fluorescent probe for detecting hydrogen peroxide according to claim 2, wherein the molar ratio of the compound Cy-NH to N, N-diisopropylethylamine is 1: (4-5).
4. The method for preparing a near-infrared two-zone fluorescent probe for detecting hydrogen peroxide as claimed in claim 2, wherein the molar ratio of the compound Cy-NH to 4-formylchlorobenzeneboronic acid is 1: (2.5-3).
5. The method for preparing a near-infrared two-zone fluorescent probe for detecting hydrogen peroxide as claimed in claim 2, wherein the amount of tetrahydrofuran added per mmol of the compound Cy-NH is 15-20 mL.
6. The method of claim 2, wherein the inert gas is nitrogen.
7. The method for preparing the near-infrared two-zone fluorescent probe for detecting hydrogen peroxide according to claim 2, wherein the stirring time in an ice-water bath is 10-20 minutes; the reaction time is 12-15 h.
8. The method for preparing a near-infrared two-zone fluorescent probe for detecting hydrogen peroxide as claimed in claim 2, wherein the purification method is silica gel chromatography.
9. The method for preparing a near-infrared two-zone fluorescent probe for detecting hydrogen peroxide as claimed in claim 8, wherein the eluent for silica gel chromatography is dichloromethane/methanol.
10. Use of the near-infrared two-zone fluorescent probe for detecting hydrogen peroxide according to claim 1 for detecting hydrogen peroxide in food.
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| WO2019217583A1 (en) * | 2018-05-09 | 2019-11-14 | Board Of Trustees Of Michigan State University | Near-infrared harvesting transparent luminescent solar concentrators with engineered stokes shift |
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| CN107207433A (en) * | 2015-01-09 | 2017-09-26 | 车医科学大学校产学协力团 | New organic compound, the near infrared fluorescent contrast agent containing the compound and the method for preparing nano particle contrast agent |
| WO2019217583A1 (en) * | 2018-05-09 | 2019-11-14 | Board Of Trustees Of Michigan State University | Near-infrared harvesting transparent luminescent solar concentrators with engineered stokes shift |
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